Phenotypic diversity of starch granules in cassava · PDF file Morphological analysis of the...

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  • Genetics and Molecular Research 16 (2): gmr16029276

    Phenotypic diversity of starch granules in cassava germplasm

    L.M. Vasconcelos1, A.C. Brito2, C.D. Carmo1, P.H.G.A. Oliveira1 and E.J. Oliveira2

    1Centro de Ciências Agrárias Ambientais e Biológicas, Universidade Federal do Recôncavo da Bahia, Cruz das Almas, BA, Brasil 2Núcleo de Recursos Genéticos e Desenvolvimento de Variedades, Embrapa Mandioca e Fruticultura, Cruz das Almas, BA, Brasil

    Corresponding author: E.J. Oliveira E-mail: eder.oliveira@embrapa.br

    Genet. Mol. Res. 16 (2): gmr16029276 Received September 14, 2016 Accepted March 8, 2017 Published April 13, 2017 DOI http://dx.doi.org/10.4238/gmr16029276

    Copyright © 2017 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution ShareAlike (CC BY-SA) 4.0 License.

    ABSTRACT. Demand for the development of cassava varieties with different native starches has guided the search for these characteristics in the germplasm of Manihot esculenta Crantz. Therefore, the objective of this study was to estimate the genetic diversity of cassava accessions for root and starch granule characteristics to guide the future industrial application of this species. Starches from 56 accessions were evaluated for the number of granules in 1 g of starch (NTG), area (AG, mm2), length (LG, mm), width (WG, mm), starch granule roundness (Round), dry matter content in the roots (DMC, %), pulp color (PulCo), and cyanogenic compounds (HCN). Images captured by light microscopy were used to determine the average phenotypic values, and these were further analyzed by principal component analysis (PCA) considering mixed data (quantitative and qualitative). Significant differences between the cassava accessions for all traits measured revealed wide variability in starch granule characteristics. Four diversity groups with better fitness for the classification of cassava accessions based on PulCo were identified, in comparison with HCN.

  • 2L.M. Vasconcelos et al.

    Genetics and Molecular Research 16 (2): gmr16029276

    Accessions with differential starch characteristics were identified, and crossings for the generation of segregating populations in order to obtain table and industry varieties have been proposed.

    Key words: Breeding; Manihot esculenta Crantz; Root; Genetic diversity; Germplasm; Starch

    INTRODUCTION

    Cassava (Manihot esculenta Crantz) is one of the most important crops of the 21st century because of its high adaptability to adverse climate and soil conditions, and its low requirement for agricultural input during production in comparison with other crops. It is used widely for human and animal subsistence and for industrial applications in food, energy, and textiles (Howeler et al., 2013).

    Cassava roots are comprised of water (70%), starch (24%), fiber (2%), protein (1%), and other substances, including minerals (3%) (Tonukari, 2004). Therefore, the most notable characteristic of cassava is its capacity to accumulate starch in its roots, the content of which varies from 70 to 90% dry matter content (Baguma, 2004). Furthermore, there is high diversity in the content, composition, physical, and chemical properties of cassava starch (Nuwamanya et al., 2010; Oliveira et al., 2015a; Sanoussi et al., 2015), resulting in its application in different industrial activities.

    Starch is synthesized in amyloplasts of plant cells, and is formed by two types of polymers: amylopectin and amylose. Amylose is present as a predominantly linear chain composed of glucose residues linked by α-1,4 bonds. In contrast, amylopectin is a branched long chain molecule, consisting of hundreds of glucose chains, α-1,4 linked by α-1,6 linkages (Hoover, 2001). Most of the known cassava varieties present starch with approximately 20-30% amylose and 70-80% amylopectin content (Taggart and Mitchell, 2009). The organization of glucose molecules in starch, including the length of the glucose chain, and the relative amylose/amylopectin ratio influence the morphology and size of the starch granules (Copeland et al., 2009).

    In general, cassava starch granules are rounded, oval, or truncated in form (circular with a flat surface on one face) and range in size from 5 to 40 µm (Ceballos et al., 2007). According to Lindeboom et al. (2004), starch granules can be grouped into four size classes: large (greater than 25 µm), average (10 to 25 µm), small (5 to 10 µm), and very small (less than 5 mm).

    Compared with cereals, cassava starch has particular properties that confer enhanced resistance to acid treatments, permitting its use in the composition of unique pastes, which makes it suitable for the production of paper, textiles, sweeteners, alcohol, and monosodium glutamate (Taylor et al., 2012). The complexity of starch biosynthesis results in large natural variability in relation to the amylose/amylopectin ratio, reflecting the diversity in granule morphology (size and shape), which is associated with different functional properties in the food industry (Wani et al., 2012). Moreover, many cassava varieties are used for starch extraction, which allows a variety of starches to be obtained, and particle distribution can vary in size, morphology, and physicochemical properties. The distribution and granule size may result in distinct starch agglomerations, influence its behavior under certain processing conditions, and consequently affect the quality of the final product (Molenda et al., 2006).

    In addition to the amylose/amylopectin ratio, molecular weight, and starch granule structure, some reports have shown that granule size can affect the composition, gelatinization paste properties, enzymatic susceptibility, crystallinity, swelling, and starch solubility (Wani et al.,

  • 3Phenotypic characterization of starch in cassava

    Genetics and Molecular Research 16 (2): gmr16029276

    2012). All of these characteristics appear to be affected by environmental and agronomic crop conditions as well as by genetic factors, resulting in substantial changes in the functional properties of the starch (Lawal et al., 2011). Although cassava starch has been extensively studied in relation to its paste properties, and for its chemical and physical composition, few studies have investigated the genetic diversity of the distribution, shape, and size of cassava starch granules. Knowledge of the genetic diversity present in germplasm banks is essential when utilizing phenotypic variations in the development of new varieties that can bring functional properties to different industrial applications (Upadhyaya et al., 2007). The aim of this study was to characterize and evaluate the genetic diversity of cassava accessions for starch granule traits, which may contribute to future industrial application and, consequently, to the greater use of genetic resources of M. esculenta.

    MATERIAL AND METHODS

    Plant material

    Fifty-six accessions of the Cassava Active Germplasm Bank at Embrapa Cassava & Fruits (Cruz das Almas, Bahia, Brazil) were collected during 11 months after planting (Table 1). The region has a hot and humid tropical climate, Aw to Am according to the Köppen classification, with average annual temperatures of 24.5°C, 80% relative humidity, and 1200 mm annual rainfall. The soil is classified as yellow Latosol dystrophic. The cassava accessions were selected based on their contrasting starch content, cyanogen compound content, and pulp color of the roots.

    Starch extraction

    Roots were harvested 11 months after planting. The selected roots were washed in water, following which 1 kg of pulp was sectioned to complete the extraction process. The selected pieces were ground in a blender using a non-cutting helix (to reduce the shearing of the starch grains and the consequent physical modification) for 90 s in a 1:1 root to cold water ratio. This process was repeated several times following 1-min pauses.

    The crushed material was filtered in voile-type fabric, and through a sieve, to analyze particle size (220 mesh) in a plastic bucket (5 L). Next, the tritured mass was washed with 3.5 L cold water. The filtrate was placed in a cold chamber at 5°C for 12 h to enable the starch to settle. The supernatant was discarded and the decanted starch was washed with 20 mL 95% alcohol to accelerate drying. The alcohol was discarded, and the starch was stored in an oven with forced air circulation at 45°C until completely dry. The dried starch was then macerated using a mortar and pestle until a finely textured powder was obtained, which was then packed in sealed plastic vacuum bags for further analysis.

    Morphological analysis of the starch granules

    To visualize the starch granules, a solution of 1 g starch and 4 g water was used to simulate the concentration of starch present in the cassava roots. Subsequently, 0.8 mL of this solution was mixed with 1.8 mL 2% iodine solution (2 g potassium iodide [KI], 0.2 g iodide [I2], and 100 mL distilled water). A 0.2-mL volume of this solution was transferred to a Neubauer chamber for observation at 400X magnification under a LEICA optical microscope (DM500, Germany). Digital images were captured using LAS EZ software, which recorded

  • 4L.M. Vasconcelos et al.

    Genetics and Molecular Research 16 (2): gmr16029276

    the edge quarters of the chamber and the center. Next, the images were processed and analyzed using ImageJ software (Schneider et al., 2012) to determine the following traits: number of granules in 1 g starch (NTG), granule area (GrAr, mm2), length of granules (GrLe, µm), width of granules (GrWi, µm), granule round